The Phylogenetic Relationships of Tachinidae
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THE PHYLOGENETIC RELATIONSHIPS OF TACHINIDAE (INSECTA: DIPTERA) WITH A FOCUS ON SUBFAMILY STRUCTURE A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By DANIEL J DAVIS B.S., Wright State University, 2010 2012 Wright State University WRIGHT STATE UNIVERSITY GRADUATE SCHOOL December 13, 2012 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Daniel J Davis ENTITLED The phylogenetic relationships of Tachinidae (Insecta: Diptera) with a focus on subfamily structure BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science _____________________________ John O. Stireman III, Ph.D. Thesis Director _____________________________ David Goldstein, Ph.D., Chair Department of Biological Sciences College of Science and Mathematics Committee on Final Examination ____________________________ John O. Stireman III, Ph.D. ____________________________ Jeffrey L. Peters, Ph.D. ____________________________ Donald F. Cipollini, Ph.D. ____________________________ Andrew Hsu, Ph.D. Dean, Graduate School ABSTRACT Davis, Daniel J. M.S., Department of Biological Sciences, Wright State University, 2012. Phylogenetic relationships of Tachinidae (Insecta: Diptera) with a focus on subfamily structure. The parasitoid flies of the Tachinidae family are an important and diverse (>10,000 species) lineage of insects. However, tachinids are not well studied partially due to their confusing classification and taxonomy. DNA sequences were obtained from twenty tribal representatives of Tachinidae, along with eight outgroups in order to phylogenetically reconstruct the superfamilial, subfamilial and tribal relationships of Tachinidae. Seven gene regions of six genes (18S, 28S, COI, CAD, Ef1a, and TPI) were sequenced for each taxon (6214 bp total). Both maximum likelihood and Bayesian methods were used to infer phylogenies. The Sarcophagidae and Oestridae were usually reconstructed as monophyletic. Calliphoridae was paraphyletic with Pollenia typically being sister to Tachinidae. The Rhinophoridae were found embedded within an otherwise monophyletic Tachinidae, a unique finding. Subfamilies of Tachinidae were generally related in a (Tachininae + Exoristinae) + (Phasiinae (Dexiinae)) manner. The problematic Tachininae genera Strongygaster (Strongygasterini) and Ceracia (Acemyini) were placed into their original subfamilies with high confidence. These findings led to a new hypothesis about a slow evolution into the parasitoid habit. iii TABLE OF CONTENTS Page INTRODUCTION………………………………………………………………….....1 BACKGROUND…………………………………………………………………...…4 Family Tachinidae……………………………………….……………….…..4 Tachinidae as Biological Control Agents…………………………….........7 Tachinid Family Relationships……………………………………….......…9 Tachinid Systematics…………………………………………………...…..11 Molecular Phylogenetics of Tachinids………………………………........13 New methods of Phylogeny Reconstruction…………………………..….15 Objectives and Hypotheses…………………………………………….…..17 MATERIALS AND METHODS…………………………………………………..…20 Experimental Design………………………………………………..………20 Laboratory Methods…………………………………………………………22 Analytical Methods…………………………………………………………..24 RESULTS…………………………………………………………………………….27 Maximum Likelihood…………………………………………………...……27 MrBayes Analyses…………………………………………………………..28 Species tree analysis in BEAST……………….…………………………..30 DISCUSSION…………………………………………………………………..……32 iv Relationships among Oestroidea………………………………………….34 Rhinophoridae………………………………………………………..……..38 Tachinid subfamily relationships……………………….………………….40 Evolution of the Parasitoid Habit…………………………………….…….44 Future Direction……………………………………………………………..46 FIGURES……………………………………………………………………..……..48 TABLES……………………………………………………………………..……….59 APPENDIX I: Settings for MrBayes…………………………………………….…64 REFERENCES………………………………………………………………..….…65 v LIST OF FIGURES Figure Page 1. Maximum likelihood results of 18S……………………………………….48 2. Maximum likelihood results of 28S……………………………………….49 3. Maximum likelihood results of COI……………………………………….50 4. Maximum likelihood results of CAD………………………………………51 5. Maximum likelihood results of Ef1α………………………………………52 6. Maximum likelihood results of TPI ……………………………………….53 7. Maximum likelihood results using GARLI ……………………………….54 8. Analysis using MrBayes using all genetic data…………………………55 9. Analysis using MrBayes with a monophyletic Tachinidae……………..56 10. Analysis using MrBayes with additional Rhinophoridae……………….57 11. Species tree analysis using BEAST……………………………………..58 vi LIST OF TABLES Table Page 1. Sample coverage of the Tachinidae……………………………………..59 2. Sample coverage of the outgroups………..….…………………..……..60 3. Evolutionary models used during analysis……………………….……..61 4. Primer Sequences………………………….….…….…….……….……..62 5. Touchdown PCR used for most amplifications…………………………63 vii INTRODUCTION Flies of the family Tachinidae (Insecta: Diptera) are ecologically and economically important due to their parasitoid lifestyle on other insects. Like other parasitoids, the larva of a tachinid develops inside of a living insect host and then kills it in order to reach adulthood. Tachinids attack a wide range of arthropod hosts including caterpillars (Lepidoptera), true bugs (Hemiptera), centipedes (Chilopoda), and spiders (Arachnida) (Wood 1987; Stireman et al., 2006). Tachinids also have a wide range of host attack strategies including laying larvae directly on the host, actively seeking out their host in the larval stage, and laying tiny eggs that the host ingests. Tachinids can be voracious parasitoids, accounting for up to 80% mortality of other insects (Boetner et al., 2000). Since the females of some species can lay up to 4,000 eggs over their lifetime (Belshaw, 1994), tachinids can be extremely effective at regulating populations of their hosts. For this reason tachinid flies have been extensively used as biological control agents against agricultural pest insects for over 100 years (Wood, 1987). Tachinids have been widely used in managed biological control programs. For example, several species of tachinids have been introduced to control the gypsy moth (Lymantria dispar). Gypsy moth larvae are serious pests that defoliate hardwood trees and were introduced from Europe to the United States (Leihold et. al, 1992). This species defoliated 26 million acres of hardwood forest in a single major outbreak (1980-1982; McManus et al., 1992). Since the establishment of several tachinid enemy species on the gypsy moth, further 1 control efforts using pesticides have not been needed (Van Driesche et al., 2010). Other examples of successful biological control with tachinids include brown tailed moth, winter moth, sugarcane borers, mole crickets, and corn earworms (Grenier, 1998). These control efforts have reduced the need for pesticides and can be economical once establishment is achieved (Myers et al., 1998). Although some control efforts using tachinids have been successful, many have been limited due to the lack of basic information about tachinids. In particular, the phylogenetic relationships of tachinids are poorly understood. The relationships among the 10,000 species of tachinids are ambiguous due to a high amount of morphological homoplasy throughout the family. This homoplasy creates identification problems and many scientists do not attempt to identify tachinids beyond the family level. This impedes managers of biological control programs when they are attempting to find suitable parasitoids for their project (Cooper et al., 2011). This is compounded by a lack of general knowledge about tachinid biology and their systematics. Systematic knowledge of tachinids is lacking due to a poor fossil record, the relative youth of the clade, morphological homoplasy, cryptic speciation, a high number of species, identification difficulties, and a lack of phylogenetic evidence (Crosskey, 1976; McAlpine, 1989; Stireman et al., 2006). However, we can now use molecular techniques to create a robust phylogeny that could fill in the knowledge gaps. A robust phylogeny also has the added benefits of bringing insight to the evolution of tachinids and their parasitoid lifestyle. 2 The focus of my research is using phylogenetics to construct a robust skeletal phylogeny for tachinids that will act as a basic framework for future research on this extremely large clade. By using phylogenetics, we can also look into the evolution of tachinids and the parasitoid lifestyle in general. The major goals of my research are: ● Use DNA sequence data to construct a robust phylogenetic framework for the family Tachinidae while emphasizing the subfamily structure. ● Place difficult taxa such as Strongygaster (Strongygasterini) and Ceracia (Acemyini) into appropriate subfamilies. ● Identify the sister-group to Tachinidae and their position within the superfamily Oestroidea. ● Use this framework to gain insight into the evolution of the parasitoid habit. These goals will be achieved by using genetic sequencing and phylogenetic analytical techniques. Through these analyses, I will provide insights into the relationships within the Tachinidae and to advance our knowledge about the evolution of the parasitoid habit. Tachinids represent a hyper-diverse lineage that may provide insight into the evolution of the parasitoid lifestyle. Tachinids are economically important as biological control agents against insect pests but a lack of basic knowledge of Tachinidae has created serious ecological problems. A robust phylogeny will clarify the relationships within tachinids as well as reveal insights into the evolution of